BÀI GIẢNG CHỦ ĐỀ MIỄN DỊCH

Immunochemical Methods in the Clinical
Laboratory

Roger L. Bertholf, Ph.D., DABCC
Chief of Clinical Chemistry & Toxicology, UFHSC/Jacksonville
Associate Professor of Pathology, University of Florida College of Medicine


Name The Antigen

ASCP/Bertholf


Early theories of antibody formation

•

Paul Ehrlich (1854-1915)
proposed that antigen
combined with pre-existing
side-chains on cell surfaces.

•

Ehrlich’s theory was the
basis for the “genetic
theory” of antibody
specificity.


The “Template” theory of antibody

formation
•

•

Karl Landsteiner (1868-1943) was
most famous for his discovery of the
A/B/O blood groups and the Rh
factor.
Established that antigenic specificity
was based on recognition of specific
molecular structures; he called these
“haptens”; formed the basis for the
“template” theory of antibody
formation.


Aminobenzene Sulphonate, a Hapten

NH2

NH2

NH2

SO3

SO3
SO3
Ortho

Meta

Para


Classification of immunochemical methods

• Particle methods
– Precipitation
• Immunodiffusion
• Immunoelectrophoresis
– Light scattering
• Nephelometry
• Turbidimetry

• Label methods
– Non-competitive
• One-site
• Two-site
– Competitive
• Heterogeneous
• Homogeneous


Properties of the antibody-antigen bond

• Non-covalent
• Reversible
• Intermolecular forces

– Coulombic interactions (hydrogen bonds)
– Hydrophobic interactions
– van der Waals (London) forces
• Clonal variation


Antibody affinity

Ab + Ag ↔ Ab • Ag

[ Ab • Ag ]
Ka =
[ Ab][ Ag ]


Precipitation of antibody/antigen
complexes
• Detection of the antibody/antigen complex depends
on precipitation
• No label is involved
• Many precipitation methods are qualitative, but
there are quantitative applications, too


Factors affecting solubility

•
•
•
•

Size
Charge
Temperature
Solvent ionic strength


Precipitate

The precipitin reaction

etc.

Zone of equivalence
Antibody/Antigen


Single radial immunodiffusion

Ag


Single radial immunodiffusion

r

r ∝ [ Ag ]


Double immunodiffusion

Örjan Ouchterlony
Developed double immunodiffusion technique in 1948


Double immunodiffusion (Ouchterlony)


Quantitative double immunodiffusion

S3

S4
P

S2

S5
S1


Electroimmunodiffusion

• Why would we want to combine immunodiffusion
with electrophoresis?
– SPEED
– Specificity
• Carl-Bertil Laurell (Lund University, Sweden)
– Laurell Technique (coagulation factors)
– “Rocket electrophoresis”

Electroimmunodiffusion

+
-


Immunoelectrophoresis

• Combines serum protein electrophoresis with
immunometric detection
– Electrophoresis provides separation
– Immunoprecipitation provides detection
• Two related applications:
– Immunoelectrophoresis
– Immunofixation electrophoresis


Immunoelectrophoresis
α-human serum
Specimen

+


Immunoelectrophoresis
-

+

P

C

γ

P

α

C

µ

P

κ

C

λ


Immunofixation electrophoresis

SPE

IgG

IgA

IgM

κ

λ


Particle methods involving soluble
complexes
• The key physical property is still size
• Measurement is based on how the large
antibody/antigen complexes interact with light
• The fundamental principle upon which the
measurement is made is light scattering
• Two analytical methods are based on light
scattering: Nephelometry and Turbidimetry


Light reflection


Molecular size and scattering

-

+

-